A number of organophosphorus (OP) compounds used by the agriculture industry and the military are highly toxic and thus hazardous to human health and harmful to the environment. For example, acetylcholinesterase-inhibiting OP compounds comprise the active ingredient of pesticides such as paraoxon as well as G-type nerve agents such as Sarin and Soman, etc., developed for chemical warfare. Thus, it is very important to be able to detoxify such OP compounds and to decontaminate surfaces and substances contaminated with these compounds.
Currently, the U.S. Army uses a nerve agent decontamination solution, DS2,which is composed (by weight) of 2% NaOH, 28% ethylene glycol monomethyl ether, and 70% diethylenetriamine (Richardson, G. A. "Development of a package decontamination system", EACR-1 310-17, U.S. Army Edgewood Arsenal Contract Report (1972), herein incorporated by reference). Although this decontamination solution is effective against nerve agents, it is quite toxic, flammable, corrosive, and can threaten the environment. Thus, there is a need for an alternative decontamination technology that is both effective and non hazardous to personnel, equipment, and/or the environment.
One approach being investigated as a potential solution to this problem is enzyme-mediated decontamination. For example, a class of enzymes known as organophosphorus acid (OPA) anhydrolases (OPAA) (EC 3.1.8.2) can catalyze the hydrolysis of a variety of OP compounds, including pesticides and fluorinated "G-type" nerve agents. (See, e.g., Landis, W. G., et al., "Discovery of multiple organofluorophosphate hydrolyzing activities in the protozoan Tetrahymena thermophila", J. Appl. Toxicol., Vol. 7, pp. 35-41, 1987; DeFrank, J. J., et al., "Screening of halophilic bacteria and alteromonas species for organophosphorus hydrolyzing enzyme activity", Chem. Biol. Interact., Vol. 87, pp. 141-148, 1993; each of which is incorporated herein by reference.) Although OPA anhydrolases are produced by both prokaryotic and eukaryotic organisms, their use for enzymatic decontamination requires an abundant supply that is readily and cheaply obtained. Thus, a recombinant microorganism that is easily grown and capable of overexpressing an OPAA having high activity against cholinesterase-inhibiting OP compounds under a variety of conditions would be very desirable.
The first OP-hydrolyzing enzyme gene to be cloned, sequenced, and expressed was that for organophosphorus hydrolase (OPH) (EC 3.1.8.1), which is encoded by the opd gene of Pseudomonas diminuta MG and the Flavobacterium sp. strain ATCC 27551. (See, e.g., McDaniel, C. S. et al., "Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase", J. Bacteriol., Vol. 170:, pp. 2306-2311, 1989, incorporated herein by reference.) The open reading frame of the opd gene contains 975 bases which encode a polypeptide of 325 amino acid residues with a molecular mass of 35 kDa. (See Mulbry, W. and Karns, J., "Parathion hydrolase gene specified by the Flavobacterium opd gene: relationship between the gene and protein", J. Bacteriol., Vol. 171, pp. 6740-6746, 1989, incorporated herein by reference.)
The inventors have recently purified another OPAA with very high OP-hydrolyzing activity from a bacteria strain, Alteromonas sp. strain JD6.5, isolated from Grantsville Warm Springs in Salt Lake City, Ut. (DeFrank J. J. and T.-C. Cheng, "Purification and properties of an organophosphorus acid anhydrolase from a halophilic bacterial isolate", J. of Bacteriol., Vol. 173, pp. 1938-1943, 1991, herein incorporated by reference.) This enzyme, OPAA-2, displays high catalytic activity at a wide pH range (6.0 to 9.5) and temperature range (10.degree. to 65.degree. C.), with optimum activity at pH 8.5 and 50.degree. C. OPAA-2 has hydrolytic activity against a number of OP compounds, including the chemical nerve agents Sarin (GB; o-isopropyl methylphosphonofluoridate), Soman (GD; o-pinacolyimethylphosphonofluoridate), and GF (o-cyclohexyl methylphosphonofluoridate), the nerve-agent-analog diisopropyl fluorophosphate (DFP), and pesticides such as paraoxon.